Whole blood and peripheral blood samples from human subjects suffering from a variety of diseases can contain both blood cells or non-blood cells (e.g., tumor cells, bacteria, etc.) suspended in a liquid medium or plasma. The blood cells include red blood cells (erythrocytes or RBCs), white blood cells (leukocytes or WBCs), and platelets. Depending on the level of maturity of the cells, red cells are further classified into nucleated RBCs (NRBCs), reticulated RBCs (reticulocytes), and mature RBCs. Mature white cells fall into one of five different “normal” categories, namely, monocytes, lymphocytes, eosinophils, neutrophils and basophils. Each of the white cell subsets can be further classified into subclasses based on their respective level of maturity, activation, lineage, function, phenotype, or abnormality. Typically, only mature cells are present in detectable amounts in peripheral blood. The red cells in a normal human outnumber the total number of white cells by about 1000: 1. Platelets, which play a role in hemostasis, are of three general types, megakaryocytes, immature reticulated platelets and mature platelets.
The differentiation and enumeration of these various types of blood cells and platelets in a patient's peripheral blood, as well as the determination of certain parameters or characteristics thereof, permit diagnosis of a variety of hematological disorders or diseases. The absolute numbers, concentrations and relative percentages of the different types of blood cells are highly indicative of the presence or absence and/or stage of certain disease states.
Current commercially available, high throughput hematology flow analyzers provide a number of measured and mathematically derived cellular indices on red blood cells, platelets and white blood cells in peripheral blood specimens. The detection and enumeration of primarily mature cell types, as well as a determination of additional cell parameters, can be accomplished by using a commercially available hematology instrument. In automatically acquiring data on each cell type, most hematology instruments use at least two discrete cell-analyzing transducers. One or more of these transducers operate to acquire data useful in differentiating and enumerating the five different types of WBCs mentioned above. Another transducer is dedicated to counting and sizing of RBCs, WBCs and platelets in a precise volume of sample. The respective outputs of the multiple transducers are processed by a central processing unit to provide an integrated cell analysis report. The respective outputs of the several transducers are correlated to provide the five-part WBC differential information. Most current hematology systems identify normal blood cell populations by examining a combination of light scatter measurements or light scatter and electrical measurements collected in sequential analyses of the same reaction mixture (i.e., an aliquot of the same sample) or from analyses of different reaction mixtures of the same sample. Conventional hematology instruments, while capable of differentiating and enumerating the vast majority of cell types and subsets normally present in a peripheral blood sample, cannot readily differentiate multiple subsets of cells in a single sample, particularly those cells that are atypical or immature. The ability to provide relevant information beyond the total white blood cell count is directly related to the inclusion of multiple analytical parameters within hematology systems. Various configurations or combinations of electrical current impedance, conductivity, light scatter, absorbance, axial light loss and fluorescence have been used to determine the five-part differential, as well as to provide flagging information for the presence of atypical cell types by using different aliquots of the same sample.
An “extended differential” measurement includes the normal 5-part differential as well as the detection and enumeration of atypical cells (e.g., cells which are considered abnormal in relation to cells in healthy human blood) and immature cells. Due to the current limitations of commercially available hematology instruments, a skilled medical technologist must perform a microscopic examination (Manual Differential) in order to obtain an extended differential analysis. A blood-smear of a sample of interest produced manually on a glass microscope slide is stained with a dye to enable all cells, including the atypical or immature cells, to be visually differentiated from each other under a microscope.
Alternatively, some blood cell types of an extended differential measurement can be detected using a conventional flow cytometer. In such an instrument, a blood sample is prepared, e.g., by either (1) mixing the sample with fluorochrome-labeled monoclonal antibodies or the like which serve to selectively “tag” certain cells of interest, or (2) mixing the sample with a fluorescent stain adapted to selectively mark cells of interest, and passed through an optical flow cell. As each cell in the sample passes through the flow cell, it is irradiated with a beam of photons adapted to excite the fluorescent material associated with the cells of interest. Fluorescent light, emitted by each of the labeled cells, and light scattered by each cell are detected and used to differentiate the cells of interest from other cells in the sample.
Flow cytometers and hematology instruments have previously been integrated into a single automated laboratory system in which blood samples are automatically advanced along a track past these different instruments. As sample-containing vials pass each instrument, a blood sample is aspirated from each vial and analyzed by the instrument. Instrument systems combining discrete hematology and flow cytometry instruments are commercially available. The requirement to correlate the respective outputs of multiple transducers in order to report certain characteristics of a cell type or subset can, under certain circumstances, be problematic, in that it introduces uncertainty in the analytical results (U.S. Pat. Nos. 5,631,165 and 5,565,499; see, e.g., Thomas et al., J. Histochem. Cytochem., 25(7): 827-835 (1977)).
Fluorescence based flow cytometry has been used to determine leukocyte lineage and state of maturation. Traditional flow cytometric analysis of multiple qualitatively distinct antigenic determinants is usually performed by employing a distinct fluorochrome for each antibody utilized in the same analysis. Usually a series of analyses are performed in order to derive clinically relevant information. This requires a separate fluorescence detector, optics and electronics for each fluorochrome used and often the incorporation of more than one laser. See, e.g., C. I. Civen et al, 1987, Internat'l. J Cell Cloning, 5:267-288; U.S. Pat. No. 5,234,816; and U.S. Pat. No. 5,137,809.
Of the technologies discussed, fluorescence based measurements have the potential to provide greater advances in hematocellular analysis. Unlike the other aforementioned technologies that take advantage of the differences in the intrinsic physical properties of cells, fluorescence detection can examine the extrinsic properties of cells through the use of probes such as fluorescent dyes, histochemical stains, and fluorescent conjugated hybridization probes or monoclonal antibodies. Fluorescence measurements have proven beneficial by providing a high degree of sensitivity and specificity through the selection of appropriate reagents. Fluorescence based flow cytometry systems have been utilized for a number of years in research environments and more recently in clinical laboratories for performing immunodeficiency analyses, DNA cell cycle analyses, and leukemia/lymphoma immunophenotyping. More recently, fluorescence measurements have been introduced on routine hematology flow systems initially for the purpose of enumerating reticulated RBCs, followed by NRBC enumeration.
A fluorescence based immuno-platelet count has also recently been announced. Of the three fluorescence measurements that have been discussed, two (reticulocyte enumeration and immuno-platelet count) are either secondary or reflex mode measurements. The only measurement that occurs as part of the leukocyte differential cycle is NRBC enumeration. This analysis is performed utilizing a nucleic acid intercalating dye (propidium iodide) and light scatter to differentiate between intact WBCs, damaged WBCs and NRBCs.
Despite the application of these technologies, the currently available analytic systems suffer from common shortcomings, including difficulty in the performance of an accurate 5-part white blood cell differential in the presence of various atypical leukocyte populations or other abnormal conditions (cellular/non cellular) that interfere with performance of the 5-part differential. In addition, the conditions that permit the detection of, or flagging for, the presence of atypical cell types suffer from high false positive or high false negative rates. These shortcomings are unacceptable because they either result in an unnecessarily high manual review rate or the failure to detect clinically significant abnormalities.
There remains a need in the art for a simple, rapid, method for determining both a comprehensive five-part differential, as well as an extended leukocyte differential, in a single analysis on either a multiparametric high throughput hematology analyzer or a specialty hematology analyzer, and compositions facilitating such methods.